PROJECT SUMMARY/ABSTRACT The propagation of electrical impulses that coordinate the rhythmic and synchronized cardiac contractions to facilitate systemic circulation is regulated by a specialized structures and cell types, the cardiac conduction system (CCS), and working cardiomyocytes in a spatial-temporally precise manner. Congenital defects of the CCS and dysregulation of CCS homeostasis can lead to CCS dysfunction, causing life threatening arrhythmias and increasing the risk of death in both children and adults. Genome Wide Association studies (GWAS) in human patients with various arrhythmias have revealed a close association between abnormal ECG and many ion channels, gap junction proteins, muscle structural proteins as well as a number of critical transcription factors that function in cardiac development and the specification, differentiation and homeostatic maintenance of the CCS, among them include the basic Helix-loop-Helix (bHLH) transcription factor Hand1 and the T-box transcription factor Tbx20. Hand1 is expressed in the early developing hearts and is essential for cardiac morphogenesis. Tbx20 is shown to be critically involved in multiple cardiogenic events and cardiac function. Interestingly, despite Hand1 and Tbx20 are not previously associated with roles in CCS development or function, GWAS analysis revealed single nucleotide polymorphisms (SNPs) within both HAND1 and TBX20 associated with prolonged QRS duration with strong linkage disequilibrium. For both HAND1 and TBX20 these SNPs are intergenic suggesting roles in transcriptional regulation of these genes. Indeed, we have identified a left ventricle (LV) Hand1 enhancer with GATA4 and T-box binding sites that resides within a conserved non-coding sequence (CNS) that lies in between the reported SNPs. Mutant mice that have harbored a gene-edited deletion of this enhancer result in a prolonged QRS. Supporting this finding, we have recently observed that Hand1 is specifically expressed within CCS structures in postnatal hearts. Transcriptional regulation of Tbx20 is expressed in multiple cardiac cell lineages. Myocardial gain- or loss-of- function studies have shown altered QRS duration and severe arrhythmia. Collectively, our overriding hypothesis is that Hand1 and Tbx20 coordinate and maintain the spatial and temporal control of the cardiac conductive system development and function via either parallel or single gene regulatory pathways. We will test this hypothesis with the following specific Aims: 1) to test the hypothesis that adult expression of Hand1 within the CCS is required for maintaining a normal QRS duration; 2) to test the hypothesis that Tbx20 functions in the development and homeostasis of the CCS as well as the maintenance of the conductive function of cardiomyocytes; 3) to test whether Hand1 and Tbx20 serves as key transcriptional regulators for CCS development and CCS physiological function in a common or different regulatory pathways.